Abstract

In vivo GITR (glucocorticoid-induced tumor necrosis factor receptor) ligation can augment T cell-mediated anti-tumor immunity, yet the underlying mechanisms of this activity, particularly its effects on CD4+CD25+foxp3+ regulatory T cells (Tregs), have not been fully elucidated. We investigated the anti-tumor activity of the agonist anti-GITR monoclonal antibody (mAb) DTA1 in a subcutaneous B16 melanoma model, in which a single DTA1 treatment (1mg i.p.) on day 4 of tumor growth induces tumor rejection in 50-60% of C57BL/6 mice. First, to identify if DTA1 was acting on effector T cells (Teffs: CD8+ and CD4+CD25-), Tregs, or both, we reconstituted RAG1-/- mice with various combinations of GITR-/- or GITR+/+ Teffs and Tregs, challenged with B16 and then treated with DTA1. GITR expression by both Teffs and Tregs was required for the maximal anti-tumor effect, whereas lack of GITR on either population led to partial anti-tumor effects. No activity was seen when both Teffs and Tregs were GITR-negative. We then explored the consequences of in vivo GITR ligation on Teffs and Tregs within normal tumor-bearing mice. First, DTA1 did not significantly alter systemic (i.e. splenic or tumor-draining LN (TDLN)) Teff or Treg frequencies, nor did Tregs within the TDLN lose their intrinsic suppressive capacity. Within the tumor, however, GITR ligation skewed the total CD8:CD4 ratio (1.3 to 1 vs. 0.7 to 1 for IgG control-treated, p<0.05), as well as Treg frequency (17% of CD4+ cells were foxp3+ vs. 40% for IgG -treated, p<0.05). This resulted in a 5-fold enhancement of the intratumoral CD8:Treg ratio (13.4 to 1 vs. 2.6 to 1 for IgG-treated, p<0.05) and enhanced tumor-specific CD8+ T cell activity, as measured by activation status and IFN-\#947; production. DTA1-treated Tregs adoptively transferred into B16-bearing mice showed decreased tumor infiltration (with normal splenic and TDLN infiltration), implying impaired tumor trafficking as one mechanism for the decreased intratumoral accumulation. In addition, immunofluorescence staining of tumors in DTA1-treated foxp3GFP mice demonstrated that the majority of Tregs that did infiltrate the tumor had lost foxp3 expression. These \#8220;former\#8221; Tregs were almost uniformly Ki67+, were not apoptotic (TUNEL-, caspase3-), and were absent in IgG-treated tumors and in DTA-1 treated TDLN, demonstrating a tumor-specific effect. Collectively, these findings demonstrate that DTA1\#8217;s efficacy is associated with a skewed intratumoral Teff:Treg ratio, derived from both impaired Treg trafficking and intratumoral reversion of Tregs into foxp3- cells which may lack suppressive function (Nat. Immunol 2007;8:277). These effects are not seen outside the tumor, making widespread loss of tolerance unlikely. These findings provide further support for the continued development of agonist anti-GITR mAb as an immunotherapeutic strategy for treating cancer.